1. Field of the Invention
The invention relates generally to signal transduction in the modulation of immunosuppression and neuroprotection and, more specifically, to the exploitation of the mechanism of immunophilin/peptidylproline cis-trans isomerase (PPIase) and Homer interaction in the development of therapeutic reagents.
2. Background Information
Many natural products are high affinity ligands to cellular proteins involved in signal transduction which are key to regulating cell growth, division and differentiation. One family of such natural products are the immunosuppressive drugs cyclosporin A (CsA), FK506 and rapamycin (Luan S., Bot Bull Acad Sin (1998) 39:217-223).
Cellular studies suggest that all three drugs suppress the immune response by blocking the activation of T lymphocytes (Schreiber S., Science (1991) 251:283-287; Kunz and Hall, Biochem Sci (1993) 18:334-338). CsA and FK506 inhibit a Ca2+-dependent signaling pathway involved in activation of T cell receptors, while rapamycin blocks a Ca2+-independent pathway required for the proliferation of T cells upon stimulation by lymphokines such as interleukin-2 (Schreiber and Crabtree, Immunol Today (1992) 13:136-141; Sigal and Dumont, Ann Rev Immunol (1992) 10:519-560). In addition to blocking T cell activation, these drugs also have inhibitory effects on signaling pathways in other systems. For example, CsA and FK506 both block the Ca2+-dependent degranulation in mast cells (Hultsch et al., 1991). Rapamycin, on the other hand, has been shown to arrest yeast and some mammalian cells at the G1 phase in the cell cycle (Heitman et al., Science (1991) 253:905-909; Bierer et al., Proc Natl Acad Sci USA (1990) 250:556-559; Dumont et al., J Immunol (1990) 144:1418-1424; Price et al., Science (1992) 257:973-977). These findings suggest that CsA, FK506 and rapamycin may target molecules that are common signaling components in different systems.
To understand the molecular mechanisms of immunosuppression by CsA, FK506, and rapamycin, the cellular receptors of these drugs have been purified and characterized (reviewed by Schreiber, 1991; Fruman et al., FASEB J (1994) 8:391-400). CsA binds to a family of receptors named cyclophilins (CyPs), and FK506 and rapamycin bind to a distinct set of receptors called FKBPs (i.e., FK506 and rapamycin-Binding Proteins). These receptors are collectively referred to as immunophilins (Schreiber, 1991).
Immunophilins have been shown to have peptidylproline cis-trans isomerase (PPIase or rotamase) activity (Harding et al., Nature (1989) 341:758-760; Fischer et al., Nature (1989) 337:476-478). Subsequent investigations have shown that the enzyme activity of both CyPs and FKBPs are competitively inhibited by binding of their specific ligands (Kofron et al., Biochemistry (1991) 30:6127-6134; Fesik et al., Science (1990) 250:1406-1409; Van Duyne et al., Science (1991) 252:839-842). However, some drug analogs can inhibit rotamase activity yet fail to suppress the immune response (Bierer et al., 1990). In particular, FK506 and rapamycin bind to exactly the same set of receptors but have different modes of action (Dumont et al., 1990; Bierer et al., 1990).
Nevertheless, it appears that the complexes formed by immunophilin and their ligands are the functional module for immunosuppression (see, e.g., Liu et al., Cell (1991) 66:807-815). For example, it has been demonstrated that the FKBP12-FK506 and CyP-CsA complexes, but not their separate components, bind to and inhibit the activity of calcineurin. Biochemical and cellular transfection studies have demonstrated that inhibition of calcineurin activity is necessary for the immunosuppressive effect of CsA and FK506 (Liu et al., Biochemistry (1992) 31:3896-3901; O'Keefe et al., Nature (1992) 357:692-694; Clipstone and Crabtree, Nature (1992) 357:695-697). In contrast, the complex formed by FKBP12 and rapamycin targets a 220 kDa protein referred to as FRAP (FKBP-Rapamycin Associated Protein) or RAFT1 (Rapamycin And FKBP12 Targets), a mammalian homologue of TOR1 and TOR2 in yeast that are involved in the signaling pathway leading to G1-S progression in the cell cycle (Kunz et al., Trends Biochem Sci (1993) 18:334-338; Brown et al., Nature (1994) 369:756-758; Sabatini et al., Cell (1994) 78:35-43).
PPIase inhibitors are used clinically as immunosuppressants, but also have neuroprotective actions (Guo et al., 2001; Snyder et al., 1998). As stated above, their immunosuppressive activity is thought to arise from secondary inhibition of the phosphatase calcineurin, however, their mechanism of neuroprotective activity remains obscure. For example, while immunophilins inhibitors have been characterized that bind to the FKBP family of proteins and inhibit rotamase activity (supra), there is no clear relationship between this activity and therapeutic efficacy. In fact, their therapeutic efficacy remains controversial and clinical trials for treatment of diseases, such as Parkinson's Disease, recently failed (Gold and Nutt, 2002). It is therefore important to understand the molecular basis for activity of PPIase agents so that simple assays can be established to screen for optimally effective compounds.
The present disclosure describes a novel mechanism of action of PPIases. Several lines of evidence indicate that the PPIase family of proteins regulate the binding of a protein described as “Homer” (see, e.g., U.S. Pat. No. 6,294,355, herein incorporated by reference, for a description of this protein) to its targets. This regulation is mediated in part by competition in binding to the Homer interacting proteins.
Homer was originally identified based on its rapid transcriptional induction in brain neurons in response to depolarizing stimuli (Brakeman et al., 1997). Homer mRNA induction does not require new protein synthesis, indicating that it is an immediate early gene (IEG). The IEG form of Homer was the founding member of a family of three mammalian genes termed Homer 1, 2 and 3 (Xiao et al., 1998; Xiao et al., 2000). The human genome also includes a Homer 2-like pseudogene.
All Homer transcripts encode an N-terminal EVH1 domain which is named based on homology to Drosophila Ena and mammalian Vasp (Xiao et al., 2000). The EVH1 domains of the three mammalian Homer genes are conserved at ˜90% identity. In the initial report that described Homer IEG (now termed Homer 1a), it was also demonstrated that Homer 1a binds the C-terminus of group 1 metabotropic glutamate receptors (Brakeman et al., 1997). In subsequent work, the site of interaction in the metabotropic receptor and a consensus sequence for binding of Homer (Tu et al., 1998) have been identified. The core consensus sequence is PPXXF (SEQ ID NO:1), termed the Homer ligand (now termed type 1 Homer ligand). The EVH1 domain interaction with the Homer ligand was further defined by a co-crystal of Homer 1 and a synthetic peptide (Beneken et al., 2000).
The co-crystal determined that the EVH1 fold is isomorphic to the plextrin homology (PH) domain (FIG. 1). The co-crystal also identified surfaces of interaction with the Homer ligand and rationalized the consensus sequence of the Homer ligand. Critical sites of contact include an association between the second proline (TPPSPF, SEQ ID NO: 2) and tryptophan W24 in the Homer 1 EVH1, and between the phenylalanine and a pocket that extends to glycine 89 of the EVH1 domain (Beneken et al., 2000). The critical contribution of these sites of interaction to the overall energetics of binding was confirmed in assays of binding that used point mutants of the EVH1 domain. The crystal also indicated that the original consensus sequence PPXXFR SEQ ID NO:21) should be modified to PXXF (SEQ ID NO:3) since the first proline is not essential for contact with the EVH1 domain. The first proline may contribute to the overall conformation of the local protein sequence. The contact at the second proline involves the amino acid backbone and not the proline side chain, so other amino acids could, in principle, substitute for proline. Additional prolines are common in natural Homer ligands and are rationalized to be important in defining the correct configuration of the ligand for binding, but not in direct contact with the EVH1 binding surface. Perhaps most importantly, the co-crystal demonstrated that the binding surface of the proline is similar to that of related EVH1 domains of Ena, Mena and Vasp, but the surface for the phenylalanine is unique to the Homer subfamily. Thus, it has been concluded that the Homer genes are a subfamily of the EVH1 family which possess unique binding surfaces and ligand sequence recognition.
All verified Homer transcripts, with the exception of Homer 1a, also encode a C-terminal coiled-coil motif (Xiao et al., 1998; Xiao et al., 2000). The coiled-coil domain mediates self-oligomerization. By forming self multimers, Homer proteins can bind to target proteins and induce them to remain in close physical association. In addition to the group 1 metabotropic glutamate receptors, Homer proteins bind to inositol trisphosphate receptor (IP3R) (Tu et al., 1998), the ryanodine receptor (RyR) (Feng et al., 2002), the scaffolding protein family termed Shank (Tu et al., 1999), and several novel gene transcripts (see, e.g., U.S. Pat. No. 6,294,355 and U.S. Application No. 2003/0027147, each herein incorporated by reference in their entirety).
Homer 1a is distinct from other Homer gene transcripts in that it is expressed as an immediate early gene and does not encode a coiled-coil domain, thus, it cannot self multimerize (Xiao et al., 1998). Based on these differences in expression dynamics and protein function, it is hypothesized that Homer 1a functions as a natural dominant negative protein to regulate the degree of coupling of Homer binding partners (Xiao et al., 2000). In physiological studies, it has been demonstrated that Homer 1a reduces coupling of mGluR to intracellular IP3R (Tu et al., 1998) and increases coupling of mGluR5 to membrane ion channels (Kammermeier et al., 2000).
One of the central uncertainties in Homer function concerns the regulation of binding. The co-crystal of Homer EVH1 and mGluR5 peptide provided an important clue in that the ligand (TPPSPF, SEQ ID NO:2) assumed a specific structural conformation. Importantly, the prolines are required to be in specific configurations that are in trans for the first two prolines and in cis for the SPF. The trans configuration is energetically preferred in most sequences, so this raises the possibility that interaction between Homer and mGluR5 (or other binding partners) may be regulated by the conformational state of the proline sequence.
All known Homer ligands include prolines, which are conformationally restricted and require the activity of cis-trans prolyl isomerases to interconvert (FIG. 2). Accordingly, the possibility was considered that PPIases play a role in regulating Homer binding to its ligands. The present invention describes a mechanism of action for signal transduction which includes Homer and PPIase interaction.
Several lines of evidence indicate that the PPIase family of proteins regulate the binding of Homer to its targets. This regulation is mediated in part by competition in binding to the Homer interacting proteins. Two protein targets that have been presently identified, which are bound by both Homer and PPIases, include mGluR5 and TrpC1. Further, it has been determined that Homer and representative PPIases bind to the same target sequence in mGluR5/TrpC1 and that there are competitive interactions that regulate their respective binding.
Immunophilins bind to PPIases and disrupt their interaction with mGluR5/TrpC1. A consequence of this disruption is that Homer can more effectively bind to mGluR5 and TrpC1. Moreover, it appears that Homer and PPIases functions are naturally interdependent; PPIases may compete for Homer binding and thereby block the formation of signaling complexes. It also appears that PPIase activity is required to place the mGluR5 sequence in the correct confirmation for Homer to bind. These findings have important implications for the development of novel PPIase agents that selectively modulate Homer signaling pathways.
Using this mechanism of action, the present invention permits implementation of simple, robust and reliable high throughput assays that can identify a novel pharmacology of PPIase inhibitors that modulate Homer signaling.